Canine heartworm disease is caused by the parasite, Dirofilaria immitis (D. immitis). This parasite is transmitted to dogs via a mosquito vector. Infective D. immitis larvae, which have developed in infected mosquitos, enter the dog through the mosquito bite wound. Upon entering the dog's circulatory system, the larvae develop and migrate to the dog's heart where they mature and breed. Dirofilaria immitis young, termed "microfilariae," migrate throughout the dog's circulatory system, where they may be ingested by a mosquito that feeds upon the infected dog. The Dirofilaria immitis life cycle is completed when the ingested microfilariae mature into infective larvae in the mosquito.
The physical presence of the heartworm parasite in the pulmonary artery and right ventricle of the canine heart, and the resulting destruction of tissue, causes respiratory and circulatory problems which can be fatal under conditions of stress or vigorous exercise. The heartworm parasite has also been shown to be the cause of focal lung, liver, eye and cutaneous lesions in man (Hamilton, R.G., et al., Exper. Parasitol., 56:298-313 (1983)).
Diagnosis of canine heartworm has, traditionally, been accomplished by the identification of D. immitis microfilariae in blood samples of dogs (Knott, J., Trans. Roy. Soc. Trop. Med. Hyg., 33: 191-196 (1939); Weiner, D. J. et al., Bull. S. E. Assoc. Biol., 17:69 (1970)). A major obstacle to accurate diagnosis is the fact that approximately 35% of heartworm-infected dogs suffer from what is known as an "occult" infection, in which no circulating microfilariae are observed. Occult infections may occur if either: (1) the heartworm infection has not progressed to the stage at which microfilariae are detectable; (2) an animal has been infected with D. immitis of only a single sex; (3) the infective D. immitis parasites are sterile; or (4) if the infection is an immune-mediated sterile infection (Rawlings, C. A. et al., J. Amer. Veter. Med. Assoc., 180:1323-1326 (1982)).
The significance of canine heartworm infection, and the importance of its accurate diagnosis even in occult cases, have led to the development of alternative methods for diagnosing canine heartworm infection. Kato, K. H. (U.S. Patent No. 4,322,495) discloses an immunoassay capable of detecting antibodies which are produced by the dog in response to the canine heartworm infection. The antibodies detected by the method of Kato comprise an unfractionated collection of immunoglobulins directed against the surface antigens of the heartworm parasite. Because this immunoassay tests for the presence of anti-heartworm antibodies, the test is useful in diagnosing both occult and overt stages of canine heartworm disease.
The diagnostic potential of this approach may be limited by the low titers of anti-heartworm antibodies which are observed in many occult heartworm infections (Scholtens, R. G., et al., Amer. J. Vet. Res., 44:861-864 (1983)). Moreover, interference from low affinity, cross-reacting canine antibodies has been found to increase the percentage of false-positive results (Gillis, J. M., et al., Amer. J. Vet. Res., 45:2289-2292 (1984)).
The deficiencies of the above described methods spurred further research aimed at developing an alternative procedure for diagnosing canine heartworm disease Weil, G. J., et al., (Amer. J. Trop. Med. Hyg., 33:425-430 (1984)) discovered that D. immitis infections could be diagnosed by the presence of parasitespecific antigens in circulating blood. Thus, according to the method of Weil, et al., an immunological reaction between a standardized antisera (containing antibodies against the D. immitis's antigens) and the sera of potentially infected dogs would indicate that a canine heartworm infection was present. Subsequent work revealed that two D. immitis' antigens (having molecular weights greater than 100,000 daltons) were the sole immunogenic proteins identified in the Weil, et al., immunoassay (Weil, G. J., Immunol., 134:1185-1191 (1985)). Hybridoma cell lines that produce monoclonal antibodies specific for these antigens were obtained and characterized by Weil, and his co-workers (Weil, G. J., et al., (1985), supra). Patent applications describing this work have been filed in the United States (Ser. No. 557,117) and Australia (Application No. 8,436,095). The immunoassay developed by Weil is currently commercialized as the FILAROCHEK.TM. CANINE HEARTWORM ANTIGEN TEST KIT of MALLINCKRODT, Inc., N.Y.
If accurately diagnosed in its early stages, heartworm disease can be successfully treated. Currently, heartworm disease is treated by administering anti-parasitic agents to infected animals (Blair, L. S., U.S. Pat. No. 4,430,329). Unfortunately, heartworm disease which has not been diagnosed in its early stages may be quite refractile to treatment. For this reason, researchers have attempted to identify compositions which could serve as a preventive vaccine. As discussed above, the life cycle of D. immitis requires both canine and mosquitoe hosts. The third larval stage (L3) of the heartworm parasite, found predominantly in mosquitoes, has been found to produce antigens which are capable of inducing an immunogenic response in dogs. (Wong, M. M., et al., Exper. Parasitol., 35:465-474 (1974)).
Wong, M. M. and co-workers irradiated L3 D. immitis larvae with X-rays, and introduced the irradiated larvae into dogs. The "vaccinated" dogs were found to produce low titers of anti-heartworm antibodies. When challenged with infective, non-irradiated L3 larvae, only 50% of the "vaccinated" animals exhibited any clinical signs of heartworm infection. The high cost of maintaining populations of live worms in experimental dogs and large colonies of vector mosquitoes, has, thus far, precluded commercial applications of the Wong "vaccine" (Wong, M. M., et al., supra).
The difficulty of producing heartworm antigens in an easily cultured microorganism was addressed by Lew K. K. (U.S. Pat. No. 4,568,639). Lew discloses the possibility of modifying an easily cultured helminth so as to enable it to produce the desired D. immitis antigens. Once such a genetically modified helminth was obtained, Lew discloses that the genes which encode the antigen could be cloned, and the antigen purified. Using his method, Lew succeeded in isolating a mutant derivative of the nematode Caenorhabditis elegans which could be used in the diagnosis of canine heartworm infections.
Thus, in summary, the prior art discloses the significance of diagnostic tests and preventive vaccines for canine heartworm disease. Diagnostic tests which are based upon the identification of either heartworm antigen, or canine antibodies directed against heartworm antigens, have been developed. The difficulty of culturing D. immitis in its canine and mosquito hosts has made the development of a commercializable and efficacious vaccine a difficult and, thus far, elusive task.